Bacterial Pathogens (1-3)

Question 1

What is a pathogen?

Answer 1

A microbe roar is able to cause disease in a plant, animal or insect

Question 2

What is pathogenicity?

Answer 2

The ability to produce disease in a host organism
→ expressed through their virulence
→ determinants of virulence: genetic, biochemical or structural features that enable it to produce a disease

Question 3

What are the underlying mechanisms of bacterial pathogenicity?

Answer 3

Ability to invade → colonisation (adherence and multiplication), ability to overcome host defence, extracellular substances that facilitate entry
→ e.g. adhesins like fimbiae

Ability to produce toxins → exotoxins: released from bacteria e.g. Cholera toxin
→ endotoxins: cell-associated e.g. LPS, capsule

Question 4

How can pathogens enter the human body?

Answer 4

Respiratory → inhalation
Gastro-intestinal → ingest
Urinary/genital tracts

via insect bites or accidental/surgical trauma to skin

Question 5

What are some examples of human primary pathogen transmission?

Answer 5

Bordetella pertussis → whooping cough, requires contact with infectious material

Neisseria gonorrhoeae → sexually transmitted, requires direct person-to-person mucosal contact - man only natural host so dies in environment

Question 6

What does the environment that a pathogen can colonise effect?

Answer 6

The environments that can be colonised by a pathogen are critical in determining its reservoirs and potential modes of transmission

Question 7

What is a psychrophile, psycotroph, mesophile and thermophile?

Answer 7

Psychrophile → microbes that grow best at low temps (optimum 10-15C)
Psychotroph → able to grow at low temps, but prefer moderate (optimum 15-30C)
Mesophile → most bacteria (optimum 30-40C)
Thermophile → optimum 45-70C

(phile = loves)

Question 8

Are bacterial growth curves symmetrical?

Answer 8

No → steady increase to optimum then crash after

Question 9

What is the acclimation phase in cold shock?

Answer 9

A downshift in temp causes inhibition of most protein synthesis - has to produce new machinery (ribosomes)
→ causes a growth lag - acclimation phase
→ a group of cold shock proteins (Csp) are dramatically induced - essential for cell growth to resume at the low temp

Question 10

What are E. coli cold shock proteins?

Answer 10

Involved in ensuring ribosome function and DNA/RNA synthesis

Class I → >10-fold induction
→ e.g. CspA family: CspA RNA chaperones, CspB/CspC RNA&DNA chaperone, RNP ribonuclease
Class II → <10-fold induction
→ e.g. RecA: recombination factor, H-NS: nucleoid-assocaited DNA-binding protein

→ conditioning ribosome for cold temps.

Question 11

What is Listeria?

Answer 11

Non-spore forming Gram +ve bacilli
→ causes listeriosis
→ serious for pregnant women and immunocompromised people
→ widespread in environment, vegetation, water4, soil, wild/domestic animals, fish

Question 12

What is unique about Listeria bacterial growth?

Answer 12

Capable of growth over a wide range of temperatures (4-40C)
→ e.g. can survive in fridge

→ can be spread through contaminated food: meats, unpasteurised milk, vegetables (coleslaw) - foods that require no further heat treatment

Question 13

What virulence factors of Listeria is involved in the invasion of mammalian cells?

Answer 13

Internalin A and B

Question 14

What virulence factors of Listeria are required for its escape from membrane vacuole?

Answer 14

LLO (listeriolysin) → pore-forming cytotoxin punches holes in membrane - surrounded by vacuole upon entry so must degrade

PI-PLC (PlcA) → an enzyme that removes charged head groups from phospholipids

double membrane vacuole:
PC-PLC (PlcB) → a phospholipase that cleaves the head group from many kinds of lipids

Question 15

What virulence factors of Listeria are required for movement through the cytoplasm (cell-cell spread)?

Answer 15

ActA → stimulates host cell actin polymerisation (in wrong place ie. surface of bacterium), actin tail forms and propels bacterium through cytoplasm
→ causes projections from the host cell enter adjacent cells

Question 16

How are Listeria virulence factors regulated?

Answer 16

PrfA → positive regulator of virulence genes
→ may repose to temp
→ DNA binding protein

Question 17

How is Listeria controlled in food?

Answer 17

Can grow and multiply at normal refrigeration temps, freezing, and relatively high cooking temps - also can grow in other food preservatives

→ controlled by: heating at 70C for 2 mins, avoiding unpasteurised milk, avoid coleslaw and deli meats, cook meat thoroughly and reheat after refrigeration

Question 18

What is Legionella pneumophila?

Answer 18

Motile, aerobic Gram -ve rod
→ cause Legionnaire’s disease - a form of bacterial pneumonia
→ normal environment: biofilm (in air con) or inside protozoa - humans accidental host

Question 19

How does pH affect growth rate?

Answer 19

Growth rate curve symmetrical around the optimum pH

Question 20

What are the terms used in relation to pH and growth?

Answer 20

Acidophiles → grow at an optimum below neutrality (>7pH)
Neutrophiles → grow best at neutral pH
Alkaliphiles → grow best under alkali conditions

Question 21

What is an obligate acidophile?

Answer 21

Require a low pH for growth
→ their membranes dissolve and cells lyse at neutrality
→ several genera of archaea

Question 22

What are obligate alkaliphiles?

Answer 22

Can grow around pH 10
→ Na+ gradient (instead of pmf = proton motive force = pH gradient and electrochemical gradient) supplies the energy

Question 23

What does the intracellular pH for most organisms need to be?

Answer 23

Intracellular pH mostly needs to stay around neutrality
→ to prevent destruction of acid or alkali labile macromolecules

anomalies: extreme acidophiles and alkaliphiles
→ their internal pH can very by several units from neutrality

Question 24

What are the problems posed by acidic conditions?

Answer 24

3 major aspects of cell function cane affected by acidic conditions
1. the capacity for nutrient acquisition and energy generation
2. cytoplasmic pH homeostasis
3. protection of proteins and DNA → critical for cell survival

Question 25

How can proteins and DNA be protected in acidic conditions?

Answer 25

Chaperone proteins and alkalisation of the periplasm

(periplasm in Gram -ve cell walls, between outer and cytoplasmic membrane)

Question 26

What is Helicobacter pylori?

Answer 26

Gram -ve curved rod, highly motile 4-7 polar flagella
Cause gastric and duodenal ulcers (associated with development of gastric cancer)
Reservoir: lining of the stomach
Main virulence factors: flagella, urease, adhesions,

Question 27

How do we know Helicobacter pylori causes ulcers?

Answer 27

Known to be associated but there was no animal model so couldn’t follow usual method to confirm Koch’s postulates
→ Barry Marshall drank culture of H. pylori - 2 weeks later had inflamed stomach

Question 28

How does H. pylori survive in the acidic pH of the stomach?

Answer 28

Organism doesn’t colonies the lumen but the mucin layer that covers the stomach
→ mucus resists diffusion of protons from stomach acid due to -ve sulphated polysaccharides

However must reach the mucin layer so needs:
→ motility
→ short term pH protection using enzyme urease

Question 29

What does the enzyme urease do?

Answer 29

Urease takes up urea (waste product) and hydrolyses it producing ammonia and CO2
→ ammonia neutralises stomach acid in its immediate vicinity
→ urease is intraceullular so exports ammonia to periplasm where pH can rise ~ pH 6

Question 30

How does Helicobacter pylori give rise to ulcers?

Answer 30

Uses urease to protect it from stomach acid during transit
→ colonises mucin layer

Products provoke inflammatory response that ultimately damage the mucosa

BabA: adhesin that recognises Lewis b antigen which binds sulphated mucin sugars on epithelial cells
NAP: neutrophil activation protein - activates neutrophils, leads to inflammation
VacA: vacuolating cytotoxin - produces large vacuoles in mammalian cells

Question 31

What is Salmonella typhimurium?

Answer 31

Characteristics: Gram-negative rod, motile
Disease: gastroenteritis, diarrhoea
Reservoir: human carriers, livestock animals, reptiles
Transmission: contaminated food
Main virulence factors: acid tolerance response, adhesins, invasion of mucosal cells, type III secretion system
Prevention: proper food handling
Treatment: antibiotics

Question 32

What is the Salmonella typhimurium acid tolerance response?

Answer 32

Allows S. typhimurium to survive in the acid environment of the stomach

Rapid exposure to low pH → rapid die-off

More gradual exposure to low pH (i.e. adapted to pH6 first) → cells can survive down to pH 3

Question 33

What cells regulate acid shock proteins?

Answer 33

Fur = the regulator of iron acquisition genes
→ Fur senses pH and iron separately
→ iron is an essential micro-nutrient

Question 34

How can pH tolerance in Gram + bacteria affect human health?

Answer 34

mutans Streptococci → causes dental caries - organisms produce acid and can survive at low pH

Listeria → causes listeriosis - drop in pH activates haemolysin permitting its escape from the phagosome, capable of surviving acid stress in food like cottage cheese, yoghurts and orange juice

Rhodococcus equi → causes broncopneumonia in horses - encounters low pH in alveolar macrophages - acid resistant

Question 35

What are the mechanisms of acid resistance in Gram positive bacteria?

Answer 35

Proton pumps → F1F2-ATP ases, GAD
Protein/DNA repair → some chaperones
Regulators → e.g. several 2 component systems (sensor + response) in Listeria
Altered metabolism → e.g. a quorum sensing system & biofilm - acid tolerance in S. mutans
Envelope alterations → e.g. S. mutans increased levels of mono-saturated and longer chain fatty acids at pH5 than pH7
Production of alkali → e.g. urease production (increased pH)

Question 36

What are obligate aerobes and anaerobes?

Answer 36

Aerobes: require O2 for growth
→ they use O2 as a final electron acceptor in aerobic respiration

Anaerobes: don’t need O2 as a nutrients (O2 is toxic - kills or inhibits growth)
→ may live by fermentation, aerobic respiration, bacterial photosynthesis or methanogensis

Question 37

What are facultative anaerobes?

Answer 37

Organisms that can switch between aerobic and anaerobic metabolism
→ no O2 - fermentation, anaerobic respiration
→ O2 - aerobic respiration

Question 38

What are aerotolerant anaerobes?

Answer 38

Bacteria with an exclusively anaerobic (fermentative) type of metabolism but are insensitive to presence of O2
→ live by fermentation alone whether of not O2 is present

Question 39

How do flavoproteins react with O2?

Answer 39

Oxidation of flavoproteins results in the formation of H2O2 (peroxide) and small quantities of O2- superoxide (an even more toxic free radical)
→ ROS can damage cells - will kill the cell if nothing is done about them

Question 40

How is the potential for lethal accumulation of superoxide prevented in aerobes?

Answer 40

The enzyme superoxide dismutase
→ all organisms which can live in the presence of O2 contain superoxide dismutase

Question 41

How is peroxide decomposed?

Answer 41

By the enzyme catalase
→ certain aerotolerant bacteria lack catalase so they use peroxidase enzymes (derive e- from NADH2 producing water)

Question 42

Why do obligate anaerobes undergo lethal oxidations by various oxygen radicals when exposed to O2?

Answer 42

They lack superoxide dismutase and catalase
→ can’t protect against ROS

Question 43

What is the action of superoxide dismutase, catalase and peroxidase?

Answer 43

Detoxify oxygen radicals

Superoxide dismutase: [O₂-] + [O₂-] (+2H+) → O₂ + H₂O₂

Peroxidase: H₂O₂ + O₂ (+ NADH + H+ → NAD+) → 2H₂O

Catalase: 2H₂O₂ → 2H₂O + O₂

Question 44

How does Clostridium spp. obtain ATP?

Answer 44

Substrate-level phosphorylation
→ most lack respiratory chain cytochromes, catalase, peroxidase and superoxide dismutase

Question 45

What is Clostridium botulinum?

Answer 45

Commonly found in soil samples and aquatic sediments
→ causative agent of botulinum food poisoning
→ releases exotoxin bolutlinum - extremely potent + can form spores
→ poorly canned foods create anaerobic environment - unskilled spores germinate and produce toxin

Question 46

What is botulism?

Answer 46

Result of ingesting bacterially produced neurotoxins
→ the toxins block the release of neurotransmitter acetylcholine
→ resulting in double vision, slurred speech, decreased saliva, general weakness
→ paralysis with accompanying respiratory failure can be fatal

Question 47

What are the types of exotoxin produced by Clostridium botulinum?

Answer 47

7 - A, B, C1, D, E, F, G
→ A, B, E, and F are the most toxic to humans (often released in inactive form, proteolytic cleavage activates them)
→ A is the most potent exotoxin known

Question 48

What is the mode of action of botulinum toxin?

Answer 48

Botulinum toxin has heavy chain (HC) and light chain (LC)
→ HC binds toxin to the presynaptic receptor - toxin enters the cell
→ disulphide bond cleaved - liberates the LC into cytoplasm and endosomal compartment
→ cleaves the SNARE proteins (involved in vesicle docking)
→ prevents fusion of acetylcholine vesicles at the cell membrane

Prevents the release of Act → no stimulus → can’t contract → flaccid paralysis

Question 49

What is Clostridium tetani?

Answer 49

Causative agent of tetanus (lockjaw)
→ result of trauma/puncture wound leading to tissue contamination
→ caused by release of single antigenic type endotoxin by C. tetani - circulates in blood and adheres to neuronal receptors
→ fixes to gangliosides - blocking release of the neurotransmitters glycine and y-amino butyric acid (GABA)
→ muscles permanently contracted

Question 50

How does the tetanus toxin work?

Answer 50

Tetanus toxin made up of a heavy chain (HC) required for cell entry and a light chain (LC) which causes disease
→ C-terminal domain of the HC binds to gangliosides, N-terminal domain allows LC to cross into cell cytoplasm
→ LC interrupts the release of neurotransmitters
→ cleaves SNARE proteins - GABA and glycine containing vesicles can’t dock thus no neurotransmitter released

Glycine usually induces muscle relaxation - constant release of Act - muscles permanently contracted

Question 51

How are hosts affected by Clostridium tetani?

Answer 51

Toxin causes tetanus
→ host usually dies from respiratory paralysis

heroin addicts are particularly susceptible

Question 52

What is Clostridium difficile?

Answer 52

Gram positive, obligately anaerobic, spore forming

Antibiotic use reduces normal microbiota → C. diff overgrows produces toxins A and B
→ A and B - large exotoxins that modify host cell membrane G proteins

Toxins cause: diarrhoea and lesions on colon surface
→ coalesce forming tissue damage - pseudomembranous colitis
→ can be rapidly fatal